This invention relates generally to novel neocartilage compositions useful as implants and as replacement tissue for damaged or defective cartilage, as model systems for studying articular cartilage disease and articular cartilage response to natural and synthetic compounds and for the isolation of cartilage derived substances useful in the biotechnology industry.
More specifically, the invention concerns neocartilage, particularly human neocartilage, having multiple layers of cells surrounded by a substantially continuous insoluble glycosaminoglycan and collagen-enriched hyaline extracellular matrix, and whose membrane phospholipids are enriched in the anti-inflammatory n-9 fatty acids, particularly 20:3 n-9 eicosatrienoic or Mead acid. Also provided are methods of producing neocartilage in vitro by growing chondrocytes in substantially serum-free growth media. The invention further relates to methods for producing conditioned growth media comprising compounds effective to enhance neocartilage formation, and for the isolation of cartilage derived substances.
Unlike most tissues, adult articular cartilage does not self-repair. Normal articular cartilage is hyaline cartilage comprising a distinctive combination of cartilage-specific collagens (types II, VI, IX, and XI) and aggregating proteoglycans (aggrecan) which give it the unique ability to withstand compressive forces.
Chondrocytes are the cartilage-specific cells which give rise to normal articular cartilage tissue growth in vivo. Adult chondrocytes, however, have generally lost their potential to reproduce and generate new cartilage in vivo, although they are responsible for maintaining tissue homeostasis.
Attempts to grow human articular cartilage using traditional cell culture methods such as growing chondrocytes on tissue-culture plastic surfaces using serum-containing growth media have proved unsuccessful. Although serum (the non-red blood cell portion of blood, clotted and spun down) is known to be a potent mitogen to chondrocytes, their culture in serum-containing growth media has been reported to result in dedifferentiation of the chondrocyte phenotype.
It is also well known in the art that growing chondrocytes in monolayers on plastic culture vessels for prolonged periods leads to loss of their spherical shape and the acquisition of an elongated fibroblastic morphology. Reginato, et al., Arthritis and Rheumatism 37: 1338-1359 (1994). Biochemical changes associated with this morphological change include loss of the articular cartilage phenotype, e.g., loss of rounded cell shape, an arrest of cartilage-specific collagen and proteoglycan synthesis, the initiation of collagen type I and III synthesis, and an increase in small non-aggregating proteoglycan synthesis. Reginato, et al., supra.
Adult human chondrocytes grown directly on tissue-culture plastic in growth media containing serum, attach to the plastic substrate and fail to deposit an insoluble matrix enriched in glycosaminoglycan. Glycosaminoglycan is the proteoglycan component essential to the physiological function of articular cartilage and is the hallmark of hyaline tissue. The extracellular matrix initially produced by methods using serum-containing growth media is not enriched in glycosaminoglycan and resorption of the matrix material occurs as the cell culture ages.
Attempts to overcome chondrocyte dedifferentiation in vitro have included culturing chondrocytes at high densities and growing them in suspension culture or on substrata that prevent cellular spreading and attachment to the tissue-culture plastic. Reginato et al. described a method of growing human fetal chondrocytes cultured on polyHEMA-coated plastic dishes in a serum-supplemented DMEM growth media. Arthritis and Rheumatism 37: 1338-1359 (1994). This method was successful at maintaining the cartilage-specific phenotype but produced only nodules resembling articular cartilage, not a continuous layer of articular cartilage tissue.
Kuettner described in U.S. Pat. No. 4,356,262, a method of producing bovine cartilaginous tissue from which an anti-invasion factor may be recovered. This method provided culturing a monolayer of chondrocytes at high densities in a suspension of serum-containing growth media in a roller bottle. This method produced nodules of tissue having an extracellular matrix, but not a continuous layer of articular cartilage tissue.
Another method that prevents chondrocyte attachment to tissue culture plastic is described in Kandel, U.S. Pat. No. 5,326,357. Kandel described methods of reconstituting bovine cartilage tissue in vitro by seeding chondrocytes on a porous tissue culture insert substrate which had been coated with type I collagen to facilitate chondrocyte attachment and growth in a serum-containing growth media. The tissue culture insert is used to separate the chondrocytes from the tissue culture plastic. This method produced a continuous cartilaginous tissue having zones of elongated and spherical chondrocytes which resemble native bovine cartilage.
Without a readily available replacement tissue, recent methods of articular cartilage repair have focused on biological resurfacing of cartilage defects with either a prosthetic device or with live chondrocytes. Methods of in vivo articular cartilage repair include transplanting chondrocytes as injectable cells or as a composition of cells embedded in a three-dimensional scaffold. These methods, like in vitro neocartilage production, have been less than completely successful. One such repair method is autogenous chondrocyte transplantation. Vacanti et al., WO 90/12603. In this method, normal chondrocytes obtained from the patient are surgically removed, cultured to increase cell number and then injected into the defective site and secured in place with a periosteal flap. Brittberg, M., et al., N. Eng. J. of Med., 331:889-895 (1994). This method requires two separate surgical procedures to complete.
Allograft transplant methods, which require a single surgery, use implants made of donor chondrocytes seeded and grown on a natural or synthetic three dimensional scaffold (Vacanti, et al., U.S. Pat. No. 5,041,138; Gendler, EP 0739631 A2). In these methods, the natural or synthetic three-dimensional scaffold is provided to give the cell culture structure and to mimic the natural extracellular matrix while the cartilage tissue is produced in vivo.
It has recently been shown, however, that neither the autogenous nor the allogenic transplant method results in consistent growth of articular cartilage in vivo, but rather results in chondrocyte dedifferentiation and formation of fibrocartilage. Because of the reduced aggrecan content of fibrocartilage, it cannot withstand the same biomechanical stresses as articular cartilage. Fibrocartilage degenerates with use, and its formation following joint repair may promote joint dysfunction and permanent disability.
In addition to the clinical need for readily available replacement tissue, healthy articular cartilage is needed for use in model systems for studying articular cartilage disease and to evaluate chondrocyte responses to growth factors, cytokines and pharmaceutical compositions.
Osteoarthritis, the most common form of arthritic disease, affects almost 16 million people in the United States alone. Osteoarthritis is characterized by the appearance of focal lesions at the cartilage surface. With advancing age and disease progression, these changes are accompanied by a marked reduction in proteoglycan content, extensive destruction of the collagen framework, a marked increase in tissue hydration, and subsequent joint dysfunction.
Osteoarthritis appears to develop within the articular cartilage of weight-bearing joints, particularly joints of the knee, hip, hand, and foot. Under normal physiological conditions, cartilage homeostasis is maintained by the resident chondrocytes. This highly specialized cell functions to synthesize, assemble, and remodel all components of cartilage extracellular matrix, including aggregating proteoglycan as well as collagens type II, VI, IX, and XI. Despite intensive research efforts to ascertain the biological basis of osteoarthritis, its development and progression remain poorly understood.
Recent studies attempting to characterize collagenolytic activity in human osteoarthritis indicate a clear need for a reliable alternative to animal models for elucidating early biological events of disease progression. Most animal tissues do not express the complexity of enzymes that have been implicated in human disease. Thus, animal models are inadequate for evaluating the efficacy of potential disease modifying agents in human osteoarthritis.
A shortage of normal articular cartilage for studying articular cartilage disease and articular cartilage response to natural and synthetic compounds exists because the only source of healthy articular cartilage currently available is from deceased adult donors which may show degenerative changes.
Among the several objects of the invention, therefore, may be noted:
the provision of novel neocartilage compositions and uses thereof;
the provision of methods of producing such novel neocartilage compositions and methods of producing conditioned growth media for use therein, and the provision of cartilage specific components useful in tissue engineering.
The neocartilage of the invention is useful, for example, as replacement tissue for damaged or defective cartilage and as a model system for studying articular cartilage disease and response to natural and synthetic compounds. Illustratively, surgical implants, or allografts, of the neocartilage were created in vitro and surgically attached to natural cartilage in animal models to repair surgically created defects.
Briefly, therefore, the present invention is directed to neocartilage characterized by one or more of the following attributes: containing membrane phospholipids enriched in Mead acid, containing membrane phospholipids depleted in linoleic or arachidonic acid, being substantially free of endothelial, bone and/or synovial cells, having a S-GAG content of at least about 400 mg/mg of OH-proline, being substantially free of type I, III and X collagen, containing a matrix substantially free of biglycan, being enriched in high molecular weight aggrecan, being produced in vitro using serum-free growth medium, being essentially free of non-cartilage material, and being characterized by having multiple layers of cells surrounded by a substantially continuous insoluble glycosaminoglycan and collagen-enriched hyaline extracellular matrix.
The present invention is further directed to methods of growing neocartilage in substantially serum-free growth media. Initially, chondrocytes are isolated and adhered to a surface using means effective to produce a monolayer cell culture. The cell culture is then grown in a substantially serum-free cell culture to produce the neocartilage of the invention and the neocartilage so produced.
The invention is also directed to a biological material containing essentially purified cartilage-specific macromolecules.
Also provided is a conditioned growth medium adapted for use in growing cartilage cell cultures which contain heparin-binding growth factors, at least one of which is a cartilage-derived morphogenetic protein (Chang et al., J. Biol Chem 269:28227-28234).
In a further aspect of the invention, a set of implant materials are provided. These materials include a reparative amount of Mead acid enriched neocartilage or neocartilage produced as described above. Also included are means for implanting and adhering the neocartilage in a target tissue locus.
In yet other aspects of the invention, methods of producing a conditioned growth media comprising compounds effective to enhance neocartilage formation and such conditioned growth media are provided, and methods of producing cartilage derived compounds and such cartilage-derived compounds, using serum-free growth media, are also provided.
In an additional aspect of the invention, a method of screening a pharmaceutical for its capacity to modulate arthritic disease is provided. In this method, the neocartilage as described herein is co-cultured with the pharmaceutical in an amount and under conditions effective for determining whether characteristic indications of arthritic modulation are observed in the neocartilage.
Other objects and features will be in part apparent and in part pointed out hereinafter.